Regulation of Sonic hedgehog (Shh) expression in the zebrafish retina [Elektronische Ressource] / presented by Saradavey Rathnam

Rathnam

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2007
• Nombre de lectures: 18
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Regulation of Sonic hedgehog (Shh) expression in the
zebrafish retina







Dissertation


submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of Doctor of Natural Sciences




Presented by
Saradavey Rathnam From Coimbatore, India
2007









Dissertation


submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences















Presnted by
Saradavey Rathnam
From Coimbatore, India
2007
















Regulation of Sonic hedgehog (Shh) expression in the
zebrafish retina



















Thesis Defense: 18.05.2007

Referees: Prof. Dr. Uwe Straehle

Prof. Dr. Jochen Wittbrodt







































The research described in this thesis is carried out at the Institute of
Toxicology and Genetics, Forschungszentrum Karlsruhe, Germany under the
supervision of Prof. Dr. Uwe Straehle.



Dedicated to

My Dear parents, Vino and Rathi




































Abreviatons pg 1
Summary pg 2-3
Introduction pg 4-34

1. The Cis regulatory network
1.1 Comparative genomics and identification of
cis-regulatory enhancers
2. Sonic hedgehog (shh) gene
2.1 Secretion
2.2 Shh signalling
2.3 Shh in vertebrate development
3. Vertebrate eye development
4. Development of zebrafish retina
4.1 Shh and retinal neurogenesis
4.2 Fgf signalling and retinal neurogenesis
5. Zebrafish Sonic hedgehog locus
6. Shh expression pattern in zebrafish
7. Scope of the project

Results pg 35-67
1. Two downstream enhancer region contribute to retina expression of the
shh:gfp transgene
2. Identification of a novel retina enhancer mediating shh:gfp expression in
the retina
3. A 300 bp region is sufficient and necessary to drive transgene expression in
the retina
4. A 40 bp core region is required for shh:gfp expression
5. Critical region of the RetE enhancer is conserved but not sufficient for
shh:gfp expression


6. The conserved 40bp region plus additional 5’ sequences are sufficient to
drive expression
7. The Ets factors Erm and Pea3 were predicted to bind to the RetE
8. Pea3 and Erm intact binding site is required for shh:gfp expression in the
GCL
9. Erm and Pea3 are expressed in the zebrafish retina
10. Erm and Pea3 act redundantly in promoting shh:gfp expression in the eye
11. Erm and Pea3 bind in-vitro to motif in M13 region
12. Erm and Pea3 also bind to weak motifs in M12 region
13. Fgfs are required for both initiation and propagation of shh:gfp wave in
zebrafish
14. Several Fgfs participate in the regulation of shh:gfp expression in the
retina
15. Shh acts in an auto-regulatory manner to drive expression in the retina

Discussion pg 68-77
1. Cis-regulatory enhancers mediate shh:gfp expression in the retina
2. Pea3 and Erm regulate RetE activity in the retina
3. Forskolin is more efficient than cyclopamine in blocking shh:gfp
4. Fgfs cooperate for the progression of the shh:gfp wave in the retina
5. Shh and Fgf act in concert for propagation of the shh:gfp wave in the retina
6. Mechanism controlling shh expression in the retina

Materials and Methods pg 78-87
Publications pg 8
Acknowledgements 89
Refrences pg 90

Abbreviations

Shh Sonichedgehog
Fgf Fibroblast growth factor
Twhh Tiggy winkle hedgehog
Ehh Echidna hedgehog
hpf hours post fertilisation
RetE retinal enhancer
GCL Ganglion Cell Layer
INL Inner Nuclear Layer
Ar-A activating region A
Ar-B egion B
Ar-C activating region C
MOs morpholinos
syu sonic-you
smu slow muscle omitted
GFP Green Fluorescent Protein
PTU Phenyl Thio Urea
Tk Thymidine kinase
HYB Hybridisation buffer
ETS Erythroblastoma Twenty Six
ERM Ets Related Molecule
PEA3 Polyoma Enhanced Virus 3

Summary
Summary

Sonic hedgehog (Shh) a vertebrate homolog of the Drosophila melanogaster gene hedgehog, is a
secreted protein that controls numerous differentiation processes during vertebrate
development. In the vertebrate retina, Shh controls neurogenesis and its expression spreads in a
wave like manner.
The shh locus has been well characterised in vertebrates and the regulatory regions
driving the expression in the notochord and the midline of the CNS were identified. In
zebrafish, shh transgenes were established that drive GFP reporter expression very similar to
that of the endogenous shh gene in the central nervous system including the retina. The cis-
regulation behind shh expression in the retina was not known. This prompted me to map the
region responsible for retina expression. I found it to be distinct from the previously described
enhancers controlling expression in the midline of the neural tube and in the notochord. This
novel regulatory region mediates expression of a GFP reporter cassette in the ganglion cell layer
(GCL) and inner nuclear layer (INL) of the retina. The expression is initiated in a ventronasal
patch and later spreads across the GCL and INL in a wave like pattern.
A deletion approach identified a 300 bp region to be sufficient and necessary for driving
expression in the retina. By a second series of mutations across this region using a linker
scanning approach, a minimal 40 bp core important for expression was identified. While one
clusters of point mutation impaired expression in both GCL and INL, another region was
mapped that affected expression exclusively in the GCL. Thus, expression in the two layers can
be separated.
Pea3 and Erm factors were predicted to bind to the retinal enhancer. In vitro protein
binding and morpholino studies prove that they are key regulatory factors responsible for
driving expression in the retina. Pea3 and Erm are factors known to act downstream of Fgf
signals. Earlier studies have demonstrated that Fgf signalling from the optic stalk is responsible
for initiation of retinal neurogenesis. Moreover, shh was shown to be required